Improved amylases can ameliorate problems inherent in certain processes, such as in conversion of vegetable starches or baking.
Crystallisation of amylopectin takes place in starch granules days after baking, which leads to increased firmness of bread and causes bread staling. When bread stales, bread loses crumb softness and crumb moisture. As a result, crumbs become less elastic, and bread develops a leathery crust.
Enzymatic hydrolysis (by amylases, for example) of amylopectin side chains can reduce crystallization and increase anti-staling. Crystallization depends upon the length of amylopectin side chains: the longer the side chains, the greater the crystallization. Most starch granules are composed of a mixture of two polymers: amylopectin and amylose, of which about 75% is amylopectin. Amylopectin is a very large, branched molecule consisting of chains of α-D-glucopyranosyl units joined by (1-4) linkages, where the chains are attached by α-D-(1-6) linkages to form branches. Amylose is a linear chain of (1-4) linked α-D-glucopyranosyl units having few α-D-(1-6) branches.
Baking of farinaceous bread products such as white bread, bread made from bolted rye flour and wheat flour and rolls is accomplished by baking the bread dough at oven temperatures in the range of from 180 to 250° C. for about 15 to 60 minutes. During the baking process a steep temperature gradient (200 to 120° C.) prevails over the outer dough layers where the crust of the baked product is developed. However, due to steam, the temperature in the crumb is only about 100° C. at the end of the baking process. Above temperatures of about 85° C., enzyme inactivation can take place and the enzyme will have no anti-staling properties. Only thermostable amylases, thus, are able to modify starch efficiently during baking.
Endoamylase activity can negatively affect the quality of the final bread product by producing a sticky or gummy crumb due to the accumulation of branched dextrins. Exo-amylase activity is preferred, because it accomplishes the desired modification of starch that leads to retardation of staling, with fewer of the negative effects associated with endo-amylase activity. Reduction of endoamylase activity can lead to greater exospecifity, which can reduce branched dextrins and produce a higher quality bread.
The conversion of vegetable starches, especially corn starch, to ethanol is a rapidly expanding industry. Maltotetraose (G4 or DP4) syrup is one of many commercially important products derived from enzymatic treatment of starch. The conversion of vegetable starches, especially cornstarch, to maltotetraose and lower sugars, such as glucose or maltose, is a rapidly expanding industry.
The current process consists of two sequential enzyme-catalyzed steps that result in the production of glucose or maltose. Yeast can then be used to ferment the glucose to ethanol.
The first enzyme-catalyzed step is starch liquefaction. Typically, a starch suspension is gelatinized by rapid heating to 85° C. or more. α-Amylases (EC 3.2.1.1) are used to degrade the viscous liquefact to maltodextrins. α-amylases are endohydrolases that catalyze the random cleavage of internal α-1,4-D glucosidic bonds. As α-amylases break down the starch, the viscosity decreases. Because liquefaction typically is conducted at high temperatures, thermostable α-amylases, such as an α-amylase from Bacillus sp., are preferred for this step.
The maltodextrins produced in this manner generally cannot be fermented by yeast to form alcohol. A second enzyme-catalyzed saccharification step thus is required to break down the maltodextrins. Glucoamylases and/or maltogenic α-amylases commonly are used to catalyze the hydrolysis of non-reducing ends of the maltodextrins formed after liquefaction, releasing D-glucose, maltose and isomaltose. Debranching enzymes, such as pullulanases, can be used to aid saccharification. Saccharification typically takes place under acidic conditions at elevated temperatures, e.g., 60° C., pH 4.3.
G4 (also referred to as DP4) syrup has a number of advantageous properties compared to sucrose syrups. For example, partially replacing sucrose with G4 syrup in a food reduces the food's sweetness without affecting its taste or flavor. G4 syrup has high moisture retention in foods and exhibits less deleterious Maillard reaction products because of its lower glucose and maltose content. G4 syrup also has higher viscosity than sucrose, thus improving food texture. G4 syrup depresses the freezing point of water less than sucrose or high fructose syrup, so G4 syrup can better control the freezing points of frozen foods. After ingestion, G4 syrup also affects osmotic pressure less than sucrose. Together, these qualities make G4 syrup ideally suited as an ingredient in foods and medical products. G4 syrup is useful in other industries, as well. For example, G4 syrup imparts gloss and can be used advantageously as a paper sizer. See, e.g., Kimura et al., “Maltotetraose, a new saccharide of tertiary property,” Starch 42: 151-57 (1990).
One of the yeasts used to produce ethanol is Saccharomyces cerevisiae. S. cerevisiae contains α-glucosidase that has been shown to utilize mono-, di-, and tri-saccharides as substrates. Yoon et al., Carbohydrate Res. 338: 1127-32 (2003). The ability of S. cerevisiae to utilize tri-saccharides can be improved by Mg2+ supplementation and over-expression of AGT1 permease (Stambuck et al., Lett. Appl. Microbiol. 43: 370-76 (2006)), over-expression of MTT1 and MTT1alt to increase maltotriose uptake (Dietvorst et al., Yeast 22: 775-88 (2005)), or expression of the maltase MAL32 on the cell surface (Dietvorst et al., Yeast 24: 27-38 (2007)). The saccharification step could be omitted altogether, if the liquefaction step produced sufficient levels of mono-, di-, or tri-saccharides and S. cerevisiae or its genetically modified variants were used for the fermentation step.
Pseudomonas saccharophila expresses a maltotetraose-forming maltotetraohydrolase (EC 3.2.1.60; G4-forming amylase; G4-amylase). The nucleotide sequence of the P. saccharophila gene encoding PS4 has been determined. Zhou et al., “Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila,” FEBS Lett. 255: 37-41 (1989); GenBank Acc. No. X16732. PS4 is expressed as a precursor protein with an N-terminal 21-residue signal peptide. The mature form of PS4, as set forth in SEQ ID NO: 10, contains 530 amino acid residues with a catalytic domain at the N terminus and a starch binding domain at the C-terminus. PS4 displays both endo- and exo-α-amylase activity. Endo-α-amylase activity is useful for decreasing the viscosity of gelatinized starch, and exo-α-amylase activity is useful for breaking down maltodextrins to smaller saccharides.
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